Roy Lysaa

Inhibition of human thiopurine methyltransferase by furosemide, bendroflumethiazide and trichlormethiazide

AbstractResults: Incubation in vitro of human recombinant and erythrocyte (RBC) thiopurine methyl transferase (TPMT) with furosemide, bendroflumethiazide and trichlormethiazide demonstrated inhibition of both enzyme preparations, with IC50 values of 170 μM, 360 μM and 1 mM, respectively. Kinetic studies revealed that the inhibition was mixed or non-competitive with regard both to the thiopurine substrate 6-mercaptopurine (6-MP) and the methyl donor S-adenosyl-L-methionine. Conclusion: Since S-methylation is a major pathway in the metabolism of thiopurines, our data point to the possibility of a clinically significant diuretic-thiopurine interaction in patients treated simultaneously with these drugs.

Thiopurine methyltransferase activity in a Korean population sample of children

Thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme that catalyzes the S‐methylation of the cytotoxic drugs 6‐mercaptopurine and azathioprine. Red blood cell (RBC) TPMT activity is subject to genetic polymorphism, and we have previously demonstrated an interethnic difference in TPMT activity. To investigate whether there was a race‐related difference in RBC TPMT activity, TPMT was measured in a Korean population sample of 309 healthy children. Mean TPMT activity in healthy Korean children was 12.4 ± 2.4 units/ml RBC, which is similar to the earlier reported TPMT activities in white populations. In contrast to the bimodal or trimodal frequency distributions of RBC TPMT activity in most other population samples, the frequency distribution histogram, the probit plot, and the Shapiro‐Wilk test supported a normal distribution of TPMT activity in this Korean population sample of healthy children. Mean RBC TPMT activity showed a tendency to decrease with age, but it was not statistically significant. No gender‐related difference in RBC TPMT activity was found.

Detection of one single mutation predicts thiopurine S-methyltransferase activity in a population of Saami in northern Norway

Thiopurine S‐methyltransferase (TPMT) activity exhibits genetic polymorphism. The purpose of this investigation was to identify TPMT mutant alleles in the Saami population as a basis of developing genotyping tests for prediction of TPMT activity. The most predominant allele in Saamis (n = 194) was the TPMT*3C allele (A719G mutation) representing 92% of the mutant alleles, with an estimated allelic frequency of 3.3%. The most frequent allele in Caucasians (n = 66) living in the same geographic area was the TPMT*3A (A719G and G460A mutations) representing 91% of the mutant alleles, with an estimated allelic frequency of 3.4%. A test for one mutation, A719G, may prospectively identify more than 90% of the Saami individuals who require reduction in thiopurine dose to avoid hematopoietic toxicity. In a Norwegian population, comprising both the major Caucasian population and a minor Saami population, the same genotyping tests (eg, tests for the A719G and G460A mutations) may be used.

Novel cGMP efflux inhibitors - Identified by virtual ligand screening (VLS) and confirmed by experimental studies

Elevated intracellular levels of cyclic guanosine monophosphate (cGMP) may induce apoptosis, and at least some cancer cells seem to escape this effect by increased efflux of cGMP, as clinical studies have shown that extracellular cGMP levels are elevated in various types of cancer. The human ATP binding cassette (ABC) transporter ABCC5 transports cGMP out of cells, and inhibition of ABCC5 may have cytotoxic effects. Sildenafil inhibits cGMP efflux by binding to ABCC5, and in order to search for potential novel ABCC5 inhibitors, we have identified sildenafil derivates using structural and computational guidance and tested them for the cGMP efflux effect. Eleven compounds from virtual ligand screening (VLS) were tested in vitro, using inside-out vesicles (IOV), for inhibition of cGMP efflux. Seven of 11 compounds predicted by VLS to bind to ABCC5 were more potent than sildenafil, and the two most potent showed K(i) of 50-100 nM.

Intraindividual variability in red blood cell thiopurine methyltransferase activity

Objective: Long-term (13 weeks) and circadian (24 hours) intraindividual variability in red blood cell (RBC) thiopurine methyltransferase (TPMT) activity in healthy subjects was studied. Methods: RBC TPMT activity was measured radiochemically.Results: The variability in RBC TPMT activity was low and was only slightly higher than the imprecision of the TPMT assay. Mean long-term intraindividual variability in RBC TPMT activity was 6.5% (CV) (n = 46). Mean intraindividual circadian variability in RBC TPMT activity was 6.4% (CV) (n = 18). Conclusions: In contrast to what has been observed in children with acute lymphoblastic leukaemia, the intra- individual variability in RBC TPMT activity in healthy subjects was low. The reported changes in baseline RBC TPMT activity in patients are probably therefore due to drugs, disease, assay variation or other, unidentified factors.

Erythrocyte fraction affects red blood cell thiopurine methyltransferase activity

Red blood cell (RBC) thiopurine methyltransferase (TPMT) metabolizes the cytotoxic drugs 6-mercaptopurine and azathioprine. RBC TPMT activity has been reported to predict clinical outcome in children with acute lymphoblastic leukaemia and in kidney transplant patients.We first suspected that the erythrocyte fraction affected the calculated TPMT activity when we examined intraindividual TPMT activities in kidney transplant recipients. We demonstrated that the erythrocyte fraction affected the calculated TPMT activity, thus causing a methodological inaccuracy. A low erythrocyte fraction gave an erroneously low TPMT activity. Mean variation of 7.0% was observed within the normal limits of the haematocrit level in healthy subjects. The slopes of the TPMT activity between erythrocyte fraction 0.1 and 0.5 were all significantly different from zero, and the activity displayed good linearity from erythrocyte fraction 0.2. There was a strong association between TPMT activity and erythrocyte fraction in a population sample of children, but not in two other population samples.We propose that the TPMT assay should be performed in lysates at a standardized erythrocyte fraction to avoid variation in activity due to the range of the haematocrit in a population.

Modulation of high affinity ATP-dependent cyclic nucleotide transporters by specific and non-specific cyclic nucleotide phosphodiesterase inhibitors

Intracellular cyclic nucleotides are eliminated by phosphodiesterases (PDEs) and by ATP Binding cassette transporters such as ABCC4 and ABCC5. PDE5 and ABCC5 have similar affinity for cGMP whereas ABCC5 has much higher affinity for cGMP compared with cAMP. Since the substrate (cGMP) is identical for these two eliminatory processes it is conceivable that various PDE inhibitors also modulate ABCC5-transport. Cyclic GMP is also transported by ABBC4 but the affinity is much lower with a Km 50-100 times higher than for that of ABBCC5. The present study aimed to determine Ki-values for specific or relative specific PDE5 inhibitors (vardenafil, tadalafil, zaprinast and dipyridamole) and the non-specific PDE inhibitors (IBMX, caffeine and theophylline) for ABCC5 and ABCC4 transport. The transport of [(3)H]-cGMP (2 µM) was concentration-dependently inhibited with the following Ki-values: vardenafil (0.62 µM), tadalafil (14.1 µM), zaprinast (0.68 µM) and dipyridamole (1.2 µM), IBMX (10 µM), caffeine (48 µM) and theophylline (69 µM). The Ki-values for the inhibition of the [(3)H]-cAMP (2 µM) transport were: vardenafil (3.4 µM), tadalafil (194 µM), zaprinast (2.8 µM), dipyridamole (5.5 µM), IBMX (16 µM), caffeine (41 µM) and theophylline (85 µM). The specificity for ABCC5 we defined as ratio between Ki-values for inhibition of [(3)H]-cGMP and [(3)H]-cAMP transport. Tadalafil showed the highest specificity (Ki-ratio: 0.073) and caffeine the lowest (Ki-ratio: 1.2).

Inhibition of PDE5A1 guanosine cyclic monophosphate (cGMP) hydrolysing activity by sildenafil analogues that inhibit cellular cGMP efflux

To determine the ability of 11 sildenafil analogues to discriminate between cyclic nucleotide phosphodiesterases (cnPDEs) and to characterise their inhibitory potencies (Ki values) of PDE5A1‐dependent guanosine cyclic monophosphate (cGMP) hydrolysis.